This invention relates to the therapeutic administration of fibrinolytic metalloproteinases, and more specifically to a method for administering such agents in vivo via localized delivery to vascular thrombi in order to effect clot lysis.
Vascular occlusions caused by blood clots such as thrombi and embolisms are serious medical maladies that can become limb or life threatening if not timely treated. Devices and methods have been developed for the treatment and removal of vascular blood clots. By way of illustration, see U.S. Pat. No. 4,447,236 (Quinn), issued May 8, 1984; U.S. Pat. No. 4,692,139 (Stiles), issued Sep. 8, 1987; U.S. Pat. No. 4,755,167 (Thistle et al.), issued Jul. 5, 1988; U.S. Pat. No. 5,167,628 (Boyles), issued Dec. 1, 1992; U.S. Pat. No. 5,222,941 (Don Michael). issued Jun. 29, 1993; U.S. Pat. No. 5,250,034 (Appling et al.), issued Oct. 5, 1993: U.S. Pat. No.5,370,653 (Cragg), issued Dec. 6, 1994; U.S. Pat. No.5,380,273 (Dubrul et al.), issued Jan. 10, 1995; U.S. Pat. No. 5,498,236 (Dubrul et al.), issued Mar. 12, 1996; U.S. Pat. No. 5,626,564 (Zhan et al.), issued May 6, 1997; U.S. Pat. No. 5,709,676 (Alt), issued Jan. 20, 1998; U.S. Pat. No. 5,865,178 (Yock), issued Feb. 2, 1999, and WO 90/07352 (published Jul. 12, 1990). Such methods and devices include infusion catheters for delivering thrombolytic or fibrinolytic agents to the blood clot and dissolving it. Infusion catheters are typically used in conjunction with enzymatically active agents that are capable of degrading the fibrin in the clot and thus effectively dissolving the clot. Such enzymes are typically referred to as xe2x80x9cthrombolyticxe2x80x9d or xe2x80x9cfibrinolyticxe2x80x9d agents.
Fibrolase is a known fibrinolytic zinc metalloproteinase that was first isolated from the venom of the southern copperhead snake (Agkistrodon contortrix contortrix). See Guan et al., Archives of Biochemistry and Biophysics, Volume 289, Number 2, pages 197-207 (1991); Randolph et al., Protein Science, Cambridge University Press (1992), pages 590-600; European Patent Application No. 0 323 722 (Valenzuela et al.), published Jul. 12, 1989; and U.S. Pat. No. 4,610,879 (Markland et al.), issued Sep. 9, 1986. Fibrolase has been shown to be fibrinolytic, and this metalloproteinase has been documented to have proteolytic activity against the fibrinogen Axcex1-chain, with reduced proteolytic cleavage of the Bxcex2-chain and no activity against the xcex3-chain of fibrinogen; Ahmed et al., Haemostasis, Volume 20, pages 147-154 (1990). Because fibrin is a principal component of blood clots, the fibrinolytic properties of fibrolase point to its potential as a clot dissolving agent for in vivo thrombolytic use; see Markland et al., Circulation, Volume 9, Number 5, pages 2448-2456 (1994), and Ahmed et al., above.
Novel Acting Thrombolytic (NAT) is a modified form of fibrolase that differs from fibrolase in that NAT contains 201 amino acids with an N-terminal sequence of SFPQR, whereas the N-terminal sequence of native fibrolase begins with EQRFPQR and is 203 amino acids in length. The amino-terminal modification was designed to prevent chemical reactions at amino acid residues that were capable of forming a variable quantity of cyclized glutamine (pyroglutamic acid) which have the potential to create lot-to-lot variations in quality and uniformity of the product. Thus, NAT can be viewed as a more stable molecule.
Despite these structural differences, NAT and fibrolase are similar with respect to enzymatic (fibrinolytic) activity. This similarity in biological activity is consistent with data indicating that the active site of the fibrolase molecule spans amino acids 139-159, as described by Manning in Toxicon, Volume 33, pages 1189-1200 (1995), and its predicted location in three-dimensional space is distant from the amino-terminus. The active site of the fibrolase and NAT molecules contains a zinc atom which is complexed by three histidine residues.
Published literature on venom-derived fibrolase has demonstrated its proteolytic activity against fibrinogen at the Lys413-Leu414 site and against the oxidized xcex2-chain of insulin at the Ala14-Leu15 site; Retzios and Markland, Thrombosis Research, Volume 74, pages 355-367 (1994); Pretzer et al., Pharmaceutical Research, Volume 8, pages 1103-1112 (1991), and Pretzer et al., Pharmaceutical Research, Volume 9, pages 870-877 (1992). NAT has also been determined to have proteolytic activity on these substrates at the same cleavage sites.
In contrast to fibrinolytic metallo-proteinases such as fibrolase and NAT, clot lysing agents such as streptokinase, urokinase and tissue-type plasminogen activator (tPA) are plasminogen activators which promote thrombolysis by activation of the endogenous fibrinolytic system. More specifically, plasminogen activators catalyze the conversion of plasminogen into plasmin, a serine protease. Plasmin is capable of cleaving fibrinogen and fibrin at arginyl-lysyl bonds, and it is through the generation of plasmin that the plasminogen activators ultimately effect fibrin degradation and clot lysis. Current commercially available thrombolytic agents are plasminogen activators, such as urokinase, streptokinase or tPA.
Unlike the plasminogen activator class of thrombolytic drugs, fibrinolytic metalloproteinases, such as fibrolase and NAT, do not rely on the endogenous fibrinolytic system (conversion of plasminogen to plasmin). Hence, this class of clot lysing agents can be distinguished from the plasminogen activators by their unique mode of action and are defined as xe2x80x9cdirectxe2x80x9d fibrinolytic agents.
Alpha2-macroglobulin is a prevalent proteinase inhibitor present in mammalian serum and is one of the largest of the serum proteins (having a molecular weight of 725 kilodaltons). The specificity of xcex12-macroglobulin for proteinases is broad, encompassing serine, cysteine, aspartic and metalloproteinase classes. The xcex12-macroglobulin molecule is a tetramer of identical subunits that are disulfide bonded in pairs with a non-covalent association of the half molecules. Thus, under reducing conditions, native xcex12-macroglobulin can be dissociated into its four monomeric subunits.
Each subunit of xcex12-macroglobulin possesses a region that is very susceptible to proteolytic cleavage (the xe2x80x9cbaitxe2x80x9d region). Proteolysis of the bait region induces a conformational change in xcex12-macroglobulin, which entraps the proteinase within the xcex12-macroglobulin molecular structure. This process is described in the literature as a xe2x80x9cvenus fly-trapxe2x80x9d interaction. Once the proteinase is entrapped, it is sterically hindered and therefore cannot access its macromolecular substrate.
In addition, a covalent bond can form between xcex12-macroglobulin and many of the proteinases that it entraps. As mentioned, entrapment of a proteinase induces a conformational change in the xcex12-macroglobulin molecule. It is presumed that upon this conformational change, a thioester bond on the interior of the xcex12-macroglobulin molecule becomes reactive and can form a covalent bond with nucleophilic residues (such as lysine) of the entrapped proteinase. Thus, within the general circulation, xcex12-macroglobulin can effectively neutralize a variety of proteinases.
Moreover, the conformational change in xcex12-macroglobulin brought about by the entrapment of a proteinase results in a form that is recognized by the reticuloendothelial system. Clearance of xcex12-macroglobulin-entrapped proteinases is generally described with half-life values in minutes and is believed to occur through the low-density lipoprotein (LDL)-receptor related protein expressed on macrophages, hepatocytes and fibroblasts. For more on xcex12-macroglobulin, see Methods in Enzymology, edited by A. J. Barrett, Academic Press, Inc., Philadelphia, (1981), pages 737-754.
Alpha2-macroglobulin is capable of forming a macromolecular complex with fibrolase, NAT and other proteinases. Unlike some proteinases that can form a dissociable complex with xcex12-macroglobulin, fibrolase and NAT are two examples of fibrinolytic metalloproteinases that form a complex which cannot be dissociated from xcex12-macroglobulin under physiologic conditions. When purified human xcex12-macroglobulin and NAT, for instance, are incubated together, formation of the complex begins in seconds and is nearly complete within a few minutes. This phenomenon shows that in vitro complex formation can be rapid and is suggestive of the potential rapidity of complex formation between xcex12-macroglobulin and NAT or other fibrinolytic metalloproteinases in vivo.
Although xcex12-macroglobulin is one of the major plasma proteins, there is nonetheless a finite quantity of xcex12-macroglobulin in the circulation that would be available to bind and neutralize a fibrinolytic metalloproteinase. The xcex12-macroglobulin binding capacity is therefore saturable. Once the xcex12-macroglobulin binding capacity has been exceeded, the concentration of unbound fibrinolytic metalloproteinase rises proportionally as additional fibrinolytic metalloproteinase is added to the sample.
The presence of xcex12-macroglobulin in the general circulation of a patient presents a challenge for the systemic (for example, intravenous) administration of fibrolase, NAT and other fibrinolytic metalloproteinases that are bound up by xcex12-macroglobulin in the general blood circulation. Unless the saturable level of innate (xcex12-macroglobulin is exceeded by the systemically administered dose of such fibrinolytic metalloproteinases, the latter will effectively be neutralized and rendered ineffective for therapeutic purposes.
In one in vivo study, conducted in rabbits, the biological effectiveness of venom-derived fibrolase was examined following systemic intravenous administration. Ahmed et al., Haemostasis, above. The dose of fibrolase used was 3.7 milligrams per kilogram, which was estimated to yield a final blood concentration of approximately 60 micrograms per milliliter in a 3.0-kilogram rabbit. This amount was chosen based on studies examining the inactivation of the enzyme in the presence of blood or plasma, presumably due to xcex12-macroglobulin (see pages 336 and 339).
In another in vivo study, the biological effect of recombinant fibrolase on clot lysis was examined in canines. Markland et al., Circulation, above. Four milligrams of this material per kilogram (of animal body weight) was infused over five minutes proximal to a pre-induced thrombus in the left carotid artery via a catheter device (see page 2450). Here again, it was noted that inactivation of fibrolase occurs in the general blood circulation presumably due to the presence of xcex12-macroglobulin (see page 2454, second column, last paragraph).
As these two studies show, the deactivating effects of xcex12-macroglobulin can be overcome by either administration or systemic dosages of fibrinolytic metalloproteinase that exceed the saturable level of innate xcex12-macroglobulin (the rabbit study) or by delivering the enzyme locally to the site of the clot (the dog study) and avoiding systemic administration. On the other hand, doses of the fibrinolytic metalloproteinase in excess of the saturable level of xcex12-macroglobulin, whether delivered systemically or locally, may exceed levels that are safe and well tolerated by the subject being treated. Notably, fibrinolytic metalloproteinases are capable of destroying not only fibrin, but they may also degrade other structural proteins and are therefore potentially toxic in vivo when present in large amounts that exceed the saturable level of xcex12-macroglobulin.
It is an object of the present invention to provide a safe and biologically effective way of using locally administered fibrinolytic metalloproteinases to lyse blood clots in vivo.
Briefly stated, this invention is a method for the treatment of a blood clot in vivo, in human subjects, by a fibrinolytic metalloproteinase, comprising locally administering a safe, biologically effective amount of the fibrinolytic metalloproteinase to the blood clot, such as by use of catheter delivery means.
By xe2x80x9csafe, biologically effectivexe2x80x9d amount is meant an amount sufficient to degrade fibrin and facilitate lysing of the clot (i.e., thrombus), while not exceeding levels significantly above the saturable level of (xcex12-macroglobulin in the circulatory system of the patient being treated (i.e., levels that may cause damage to blood vessel walls). Typically, this amount will be in the range between 0.025 and 1.7 milligrams per kilogram of body weight for the human subject being treated, as determined from a study conducted with blood samples from human subjects that have been studied for in vitro xcex12-macroglobulin content and binding capacity. From the in vitro results of this study, it has been possible to define the saturable level in vivo of xcex12-macroglobulin for all practical purposes, thus enabling the delineation of a biologically effective amount that takes into account not only the minimum level of fibrinolytic metalloproteinase required for biological effectiveness, but also the maximum level for well tolerated administration. This study is described in detail further below among the Examples.
The method of this invention is applicable for in vivo therapeutic use in the treatment of stationary fibrin clots located in either native arterial or venous blood vessels, or in synthetic arterial or venous grafts, in humans.
The terms xe2x80x9clocallyxe2x80x9d or xe2x80x9clocalizedxe2x80x9d as applied to the form of delivery of the fibrinolytic metalloproteinase herein refer to intra-arterial or intravenous administration either directly to the blood clot itself (i.e., intrathrombus) or in close proximity to the clot (either proximal or distal relative to blood flow) and near enough for the majority of the fibrinolytic metalloproteinase to be absorbed by the clot.
The term xe2x80x9ccatheter delivery meansxe2x80x9d is employed herein in the conventional sense of referring to a tubular medical device for insertion into canals, vessels, passageways or body cavities for the purpose of injecting or withdrawing fluids or to keep a passageway open. In general, such means will typically comprise an elongated flexible catheter body containing one or more interior passageways (or xe2x80x9clumensxe2x80x9d); a proximal portion which allows material (i.e., clot lysing agent) to be introduced into the catheter body and to flow through the lumen; a distal portion optionally having a tapered end; and multiple exit ports at or near the end of the distal portion to permit material to exit the catheter in response to applied pressure.
The method of this invention is illustrated further below with respect to peripheral artery occlusion (PAO). PAO finds its origins in peripheral vascular disease due to atherosclerosis. The symptoms develop slowly over many years as atherosclerosis progresses, with a critical ischemic level being reached in about 15 to 20% of patients with lower extremity disease. Medical therapy is limited and predominantly aimed at prevention or risk reduction using medications such as lipid-lowering or antiplatelet agents, smoke cessation programs and physical exercise. Jackson and Clagett, Chest, Volume 114, pages 666S-682S (1998).
The clinical manifestations of peripheral vascular disease may include acute occurrences of limb-threatening ischemia or the presence of more chronic evidence of vascular disease (i.e., intermittent claudications). Outside of the aforementioned preventive measures, chronic PAO is typically not treated until the onset of severe lifestyle limitation or limb-threatening ischemia. Depending on the vessel segment affected and the extent of occlusion, available medical interventions include percutaneous transluminal angioplasty, surgical revascularization, and thrombolysis. Studies have shown that the intra-arterial infusion of clot lysing agents, particularly in the early stages of occlusion, can avoid the need for surgical intervention. As demonstrated in the Rochester trial, which compared thrombolysis with the plaminogen activator urokinase to surgery in the treatment of acute PAO (Ouriel et al., Journal of Vascular Surgery, 1994, Volume 19, pages 1021-1030), approximately 33 percent of patients in the thrombolysis arm of the study were successfully treated with medical intervention only, thereby avoiding a more invasive procedure. In contrast, 98 percent of patients in the operating arm were subjected to an endovascular or surgical procedure.
Other medical disorders involving occlusive blood clots can be effectively treated in a similar manner by the present method, including, but not limited to, acute myocardial infarction, ischemic stroke, deep venous thrombosis and pulmonary embolism. The method of this invention can also be employed to dissolve clots which occur with chronically implanted medical devices such as indwelling catheters and hemodialysis access grafts.